Investigation of Rab35 and EPI64C continue to be important to our research objectives, given our finding that EPI64C and Rab35 regulate a recycling pathway in T-cells and contribute to IS formation, most likely by participating in TCR transport to the immunological synapse. Because of the singular usefulness of knockout mice in understanding key biological functions of gene, we have been expending much of our effort in this direction. We obtained Rab35 ES cells from Bay Genomics, generated chimeras, and demonstrated that Rab35 knockout is embryonic lethal. Therefore, we have undertaken creation conditional knockout mice for each of these genes. We have designed and generated gene targeting constructs suitable for making conditional knockout mice, generated and screen ES cell clones and generated chimeras. Rab35 chimeras have been born and await breeding. Problems have been encountered with the EPI64C chimeras so that they behave as constitutive knockouts and knockout of EPI64C proves to be embryonic lethal. Problems with the outcome of the targeting are being analyzed and will be solved to create a conditional knockout Roles of other molecules in T-cell recognition are being analyzed in their respective projects, ERM in BC 010995, and NHERF1 and Myo1G in BC 010993.